Mutations were introduced at all positions of the internal conserved sequence (ICS) and at three positions in the 5' junction sequence of a Saccharomyces cerevisiae actin intron contained within an actin-thymidine kinase fusion gene. Stage I of splicing is reduced by changes at all these positions. C or A replacement at the fifth nucleotide of the 5' sequence reduces the fidelity of RNA cleavage at the 5' exon-intron junction and results in an accumulation of aberrant lariat intermediate. Stage II of splicing is affected by changes in the first and second residues of the 5' sequence and in the penultimate position of the ICS. An A to G transition at the branch point of the ICS causes a major accumulation of lariat intermediate.